The concept of using attenuated pathogenic bacteria as a vaccine component has been widely disclosed and practiced. The methods for obtaining such attenuated bacteria involve selecting random mutants by chemically- or irradiation- induced mutants or producing recombinant bacteria of pathogenic origin in which a gene involved in some metabolic pathway of the bacteria has been inactivated by genetic engineering.
Straley et al. (1984) have studied the survival of mutant avirulent Yersinia pestis that carry defects in one or several metabolic pathways.
Noriega et al. (1994) have genetically engineered an oral Shigella strain for use as a vaccine prototype by introducing deletions in a gene (aroA) coding for a protein involved in the metabolic pathway of the aromatic amino acids and have demonstrated that the defective recombinant resultant Shigella strains were able to induce protective antibodies against the wild pathogen.
Substantial work has been also done using Salmonella as a model. See, for example the reports of Hoiseth et al. (1981), Levine et al. (1987), Oyston et al. (1995) and Curtiss (1990).
However, similar work has not yet been done for Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), which infects one-third of the world's population and kills 3 million people each year. TB is the largest cause of death in the world caused by a single infectious organism (Bloom and Murray, 1992). According to the WHO, more people died from TB in 1995 than in any other year in history. Worldwide, 60 million people suffer from active TB and annually 7 million new cases arise (Dolin et al., 1994). It has been estimated that, at current rates, up to half a billion people will suffer from TB in the next 50 years.
Efficient chemotherapy exists but requires lengthy and expensive treatments, making its widespread use and control difficult to achieve in developing countries. Prophylactic vaccination against tuberculosis with the attenuated stain of bovine mycobacteria, BCG (Mycobacterium bovis Bacillus Calmette--Guerin), is more cost effective and has indeed been employed worldwide. Although BCG vaccination has provided protection against tuberculosis in certain populations, the variation in efficacy of this vaccine in different field trials and its modest protective effect against the adult form of the disease (estimated by meta-analysis to be about 50%) (Colditz et al., 1994) are points of major concern. These considerations have led the WHO to place TB control efforts, notably through the development of new vaccines, among its top priorities.
However, despite its importance, the genetic determinants of M. tuberculosis virulence remain poorly characterized. In the recent years, considerable efforts have been made towards the identification of individual mycobacterial antigens involved in the immune response to tuberculosis (Young et al., 1992) with the aim of developing subunit vaccines. The observation that only live vaccines confer high levels of protective immunity against tuberculosis (Weiss & Dubos, 1955; Orme, 1988), in addition to the fact M. tuberculosis short-term culture filtrates containing proteins secreted by actively replicating bacteria were shown to protect mice against a subsequent challenge with the virulent strain (Andersen, 1994), suggested that proteins secreted by M. tuberculosis might be good candidates for the design of subunit vaccines. Indeed, antigens such as ESAT 6 (Andersen et al., 1995), mpt64 (Haslov et al., 1995), the antigen from the 45/47 kDa complex (Romain et al., 1993) and the components of the antigen 85 complex (Wiker & Harboe, 1992) were identified as powerful immunogens eliciting delayed-type hypersensitivity (Haslov et al., 1995; Romain et al., 1993), antibody responses (Romain et al., 1993; Wiker & Harboe, 1992) and the proliferation of T-lymphocyte populations responsible for long-lived immunity (Andersen et al., 1995) in guinea pigs or mice. Some of these antigens showed protective efficacy in the mouse model of tuberculosis when used as DNA vaccines (Huygen et al., 1996).
An alternative strategy to develop novel vaccines consists of constructing mutant strains of mycobacteria that are rationally attenuated. The recent development of genetic tools for performing site-specific (Pelicic et al., 1997) and random-site mutagenesis (Pelicic et al., 1997; Bardarov et al., 1997) in organisms from the Mycobacterium tuberculosis complex now renders feasible the accomplishment of such a goal. Live vaccines should have advantages over subunit vaccines in that i) they represent a greater pool of antigens which presumably should cover a wider range of T-cell repertoires, and ii) they are generally more cost effective to produce. Moreover, attenuated mutants of M. tuberculosis should express homologous protective antigens which the BCG strains lack, and, thus, elicit a more specific and stronger protective immune response against virulence challenge. In support of this hypothesis, the molecular analysis by Mahairas and collaborators (1996) of genetic differences between M. bovis BCG and its virulent counterparts M. bovis and M. tuberculosis clearly established the existence of regions of deletion in the genome of BCG (representing about 30 kb in all), some of which contain the ORFs encoding the highly immunogenic ESAT 6 and mpt64 antigens (the latter being absent from certain BCG strains only (Oettinger & Andersen, 1994)).
Among attenuated strains of intracellular bacterial pathogens, auxotrophic mutants carrying defects in the shikimate or the purine biosynthetic pathways were shown to be of particular interest as potential live vaccines candidates because they are attenuated in vivo and have the ability to retain their immunogenicity. Some Salmonella, Yersinia and Corynebacteria purine and aromatic amino acid auxotrophs have LD50s in mice 3 to 6 log.sub.10 higher than that of the wild type (Hoiseth & Stocker, 1981; O'Callaghan et al., 1988; McFarland & Stocker, 1987; Bowe et al., 1989; Simmons et al., 1997). These Salmonella auxotrophs as well as a Brucella purE deficient mutant are, however, able to persist several weeks in mice (Crawford et al., 1996; O'Callaghan et al., 1988) and the aroA and purA mutants of Salmonella typhimurium are able to induce protective immunity in mice against a challenge with the virulent strain (Hoiseth & Stocker, 1981; McFarland & Stocker, 1987).
However, the extreme difficulty in creating defined mutants of M. tuberculosis, either by allelic exchange or transposon mutagenesis, has prevented identification of its virulence factors following Koch's postulates (Falkow, 1988; Jacobs, 1992). Rather, alternative genetic strategies have been used, including complementation of non-pathogenic bacteria (Arruda et al, 1993) and spontaneous avirulent mutants with libraries of virulent M. tuberculosis (Pascopella et al. 1994) or M. bovis (Collins et al., 1995) chromosomal DNA. Although these studies have identified genes required for entry into epithelial cells and conferring a growth advantage in vivo, the great majority of the mycobacterial genes involved in virulence remain unknown. Developing efficient mutagenesis systems is thus a top priority for mycobacterial genetics.
One method for creating mutants is allelic exchange mutagenesis. Recently, low-frequency allelic exchange was demonstrated in bacteria of the M. tuberculosis complex using a suicide delivery vector (Reyrat et al., 1995; Azad et al., 1996), and new protocols allowing easier detection of allelic exchange mutants have also been developed Norman et al., 1995; Balasubramamian et al., 1996; Pelicic et al., FEMS Microbiol. Lett. 1996). However, detection of very rare allelic exchange events is hindered by low transformation efficiencies and high frequencies of illegitimate recombination. Thus, many mycobacterial genes still remain refractory to allelic exchange by available technology.
Clearly, the allelic exchange mutagenesis system requires the design of more efficient methods. The problems encountered can be circumvented by using a replicative delivery vector which is efficiently lost under certain conditions. Allowing the introduced delivery vector to replicate avoids the problems arising from low transformation efficiencies. Then, under counter-selective conditions, clones that still contain the vector are eliminated, allowing the detection of very rare genetic events. One such system has recently been developed. Using a conditionally replicative vector which is efficiently lost at 39.degree. C. in M. smegmatis, the first mycobacterial insertional mutant libraries were constructed in this fast-growing model strain (Guilhot et al., 1994). However, the thermosensitive vectors used are only weakly thermosensitive in slow-growing mycobacteria of the M. tuberculosis complex and therefore cannot be used in these species for allelic exchange mutagenesis (unpublished data).
To date, it has not been possible to inactivate any specific gene of a mycobacterium strain via allelic exchange due to the absence of an efficient positive counter-selective marker gene that allows for selection of recombinant mycobacteria carrying a defective metabolic pathway gene. Thus, it has not previously been possible to generate a mycobacterium strain with an inactivated gene, particularly a gene involved in a metabolic pathway of the pathogenic mycobacteria, such that the defective strain is able to replicate only at a very low level in the host but does persist in the host long enough to allow the induction of an immune response. Nor has it been possible to produce an attenuated recombinant mycobacterium strain that is incapable of inducing disease in a host to which it has been administered.